Methods for producing polypeptides in Aspergillus mutant cells

ABSTRACT

The present invention relates to methods for producing a polypeptide of interest by (a) cultivating a mutant of a parent  Aspergillus  cell, wherein (i) the mutant comprises a first nucleic acid sequence encoding the polypeptide and a second nucleic acid sequence comprising a modification of at least one of the genes responsible for the biosynthesis or secretion of at least one toxin, and (ii) the mutant produces less of the toxin than the parent  Aspergillus  cell when cultured under the same conditions; and (b) isolating the polypeptide from the culture medium. Also, mutants of  Aspergillus  cells are provided, as well as methods for obtaining the mutant cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/099,704 filed Mar. 15, 2002, which is a divisional of U.S.application Ser. No. 09/472,364 filed Dec. 23, 1999, now U.S. Pat. No.6,383,781, which claims priority or the benefit under 35 U.S.C. 119, ofDanish application nos. PA 1998 01726 and PA 1999 00745 filed Dec. 23,1998 and May 27, 1999, respectively, and U.S. provisional applicationNos. 60/117,396 and 60/139,593 filed Jan. 27, 1999 and Jun. 17, 1999,respectively, the contents of which are fully incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to methods for producing polypeptides ofinterest in toxin-deficient Aspergillus mutant cells. The presentinvention also relates to mutants of Aspergillus cells and to methodsfor obtaining said mutant cells.

BACKGROUND OF THE INVENTION

The use of recombinant host cells in the expression of heterologouspolypeptides has in recent years greatly simplified the production oflarge quantities of commercially valuable polypeptides, such asindustrially important enzymes and secondary metabolites, whichotherwise are obtainable only at lower quantities or by purificationfrom their native sources. Currently, there is a varied selection ofexpression systems from which to choose for the production of any givenpolypeptide, including eubacterial and eukaryotic hosts. The selectionof an appropriate expression system often depends not only on theability of the host cell to produce adequate yields of the polypeptidewith the desired composition and conformation, but, to a large extent,may also be governed by the intended end use of the protein.

One problem encountered in connection with the use of certain hostsystems is the production of mycotoxins. A number of fungi, which areused as host cells in the production of polypeptides of interest,possess genes encoding enzymes involved in the biosynthesis of varioustoxins. For example, cyclopiazonic acid, kojic acid, 3-nitropropionicacid and aflatoxins are known toxins, which are formed in, e.g.,Aspergillus flavus. Similarly, trichothecenes are formed in a number offungi, e.g., in Fusarium sp. such as Fusarium venenatum and inTrichoderma. A detailed overview of the formation of toxins in differentfungi can be found in Handbook of Toxic Fungal Metabolites, Richard JCole and Richard H. Cox, Academic Press, 1981.

The formation of such toxins during the fermentation of the polypeptidesof interest is highly undesirable as they may present a health hazard tooperators, customers and the environment.

Consequently, a lot of effort is spent ensuring that such toxins are notformed under the conditions used in the relevant productions is inlevels considered to affect the health. This is mainly done by anextensive analytical program where the toxins are analyzed directly andby bioassays and/or feeding studies. In many cases these extensiveprograms are carried out on every single production batch affecting bothproduction costs and the time before the products can be sold.

Cyclopiazonic acid (hereinafter also referred to as “CPA”) is a weakacid (pK_(a): 3.5) and precipitates under acidic conditions. It formsmetal chelates, which can be split by dilute acid. It is quite toxicleading among other things to degenerative changes and necrosis in manyorgans, and selectively inhibits Ca²⁺-ATPase. CPA is produced in analpha- and beta-form, the beta-form being a precursor for thealpha-form. CPA is produced by Aspergilli but also by other fungi, suchas Penicilli.

Kojic acid (hereinafter also referred to as “KA”) is produced by a largenumber of Aspergilli but also by other fungi, such as Penicilli, andeven by some bacteria. It is weakly alkaline (pK_(a): 7.9; phenolicgroup), and forms complexes with many metal ions. It has antimicrobialactivity and is weakly toxic to animals. It is a precursor for a numberof synthetic compounds like insecticides, dyes, etc.

3-Nitropropionic acid (hereinafter also referred to as “3-NPA”) is anatural nitro compound. It is produced by some fungi, especiallyAspergilli (A. flavus, A. wentii) and Penicilli (P. atroventum). It hasbeen reported in a few bacteria. The acid or its esters are also foundin some plants. It is rather toxic in itself leading to, e.g., anemia.Also, it may be partly converted to another toxic compound nitrite inthe gastrointestinal tract. 3-Nitropropionic acid affects Krebs cycle byinhibiting succinate dehydrogenase irreversibly and isocitrate lyase,fumarase and aspartase reversibly.

Aflatoxins are extremely biologically active, secondary metabolitesproduced by the fungi Aspergillus flavus Link ex. Fries and Aspergillusparasiticus Speare; see R. W. Detroy et al. “Aflatoxin and relatedcompounds”, In Microbial Toxins, Vol. 6 (A. Ciegler, S. Kadis, and S. J.Aj1, Eds.), Academic, New York, 1971, pp. 3-178. The major aflatoxinsare B₁, B₂, G₁, and G₂. The metabolites, particularly aflatoxin B₁, arenot only toxic to animals as well as humans but are also the mostcarcinogenic of all known natural compounds.

Malformins and ochratoxins are produced by A. niger.

By eliminating or reducing the ability of the host organisms to producetoxins both the regulatory approval procedure will be much simpler andtime and money can be saved in the production phase as the analyticalprogram can be reduced.

Currently, there is a need for toxin-deficient Aspergillus mutant cells(i.e., safe organisms preferably classified as GRAS), which are suitablefor producing polypeptides of interest in an efficient and economicalway. The present invention satisfies this need by providing theproduction of polypeptides of interest using toxin-deficient Aspergillusmutant host cells and by providing a method to construct such mutanthost cells. There is also a need for providing Aspergillus mutant cells,which in parent form harbor (a) toxin gene(s), which is(are) notexpressed, i.e., silent gene(s).

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention a method isprovided for producing a polypeptide of interest by an Aspergillusmutant host cell, which comprises (a) cultivating a mutant of a parentAspergillus cell, wherein (i) the mutant comprises a first nucleic acidsequence encoding the polypeptide and a second nucleic acid sequencecomprising a modification of at least one of the genes responsible forthe biosynthesis or secretion of at least one toxin, and (ii) the mutantproduces less of the toxin than the parent Aspergillus cell whencultured under the same conditions; and (b) isolating the polypeptidefrom the culture medium.

In a preferred embodiment of the invention, the mutant Aspergillus cellsproduce at least about 90% less of the toxin than the parent cell whencultured under the same conditions. Preferably, the mutant produces lessof a one or more of cyclopiazonic acid, kojic acid, 3-nitropropionicacid and aflatoxin than the parent Aspergillus cell when cultured underthe same conditions.

According to another embodiment of the present invention toxin-deficientAspergillus mutant host cells are provided useful for the production ofa heterologous polypeptide of interest, which cell has is beengenetically modified in order to produce less of at least one toxin ascompared to an Aspergillus parental cell, when cultured under the sameconditions.

The Aspergillus mutant cells according to the invention are preferablyselected from the group of A. oryzae, A. aculeatus, A. nidulans, A.ficuum, A. flavus, A. foetidus, A. soja, A. sake, A. niger, A.japonicus, A. parasiticus, and A. phoenicus.

In a further embodiment of the present invention a method is providedfor obtaining a toxin-deficient Aspergillus mutant host cell, whichcomprises (a) introducing into an Aspergillus parent host cell a firstnucleic acid sequence encoding a polypeptide of interest and a secondnucleic acid sequence comprising a modification of at least one of thegenes responsible for the biosynthesis or secretion of at least onetoxin; and (b) identifying the mutant from step (a) comprising thenucleic acid sequences.

In an aspect the invention relates to Aspergillus mutant cells, suitablefor the expression of heterologous polypeptides, wherein one or moresilent toxin genes have been eliminate.

These and other embodiments will be outlined in further detail in thedescription, which will follow hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the genomic restriction mapping of the Aspergillus oryzaeDCAT-S gene carried out with the restriction enzymes EcoRI, SalI, BbuI,XhoI and XbaI, using the 1 kb ³²P-labelled DNA BglII fragment frompJaL499 containing the DCAT-S gene as a probe.

FIG. 2 shows the construction of the disruption plasmid p(DCAT-S-pyrG).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

For the purpose of the present application, the following terms aredefined for a better understanding of the invention.

The term “vector(s)” means plasmid, cosmid, phage or any other vehicleto allow insertion, propagation and expression of a gene or DNA sequenceencoding a polypeptide of interest including precursor forms thereof.

The term “host(s)” means any cell that will allow expression of apolypeptide of interest including precursor forms thereof.

The term “transformation” means incorporation permitting expression ofheterologous DNA sequences by a cell.

The term “mutant” host (or strain) means a genetically modified strain,which includes both transformants and mutants of a wild type strain.

The term “toxin” means a metabolite of fungi with phytotoxic, zootoxicand antibiotic activity. The term “mycotoxin” is normally defined as asecondary metabolite produced by a fungus with the potential to cause anadverse health effect in humans and animals at levels of exposure. Inthe present context the terms “toxin” and “mycotoxin” may be usedinterchangeably.

The term “toxin deficient” as used about a mutant cell of the inventionmeans that the mutant has an incomplete toxin production as compared toits parent strain, i.e., that the mutant produces less of at least oneand preferably more than one toxins than the parent cell.

The term “same conditions” as used in step a) of the method of theinvention relates the toxin production of the mutant to that of theparent cell and is intended to indicate that similar conditions, e.g.,with respect to pH, temperature, oxygen, etc. are used for thefermentation of the mutant and the parent strain. The parent and mutantcells may be compared with regard to production of the toxin(s) inquestion under conditions conducive for the production of a polypeptideof interest or under conditions conducive for the production of thetoxin(s).

Host cells

The present invention provides mutants of Aspergillus host cells usefulfor the expression of polypeptides of interest, wherein the cells havebeen genetically modified in order to express significantly reducedlevels of one or more toxins in comparison to a parental cell. The hostcell is derived from the parental cell, which may be a wild type cell.

The host strains may be any Aspergillus host cell conventionally usedfor the expression of polypeptides of interest.

In a preferred embodiment, the Aspergillus host cell useful for theproduction of a polypeptide of interest is selected from the groupconsisting of the Aspergillus subgroups Eurotium (e.g., represented bythe species A. restrictus), Chaetosartorya (e.g., represented by thespecies A. cremeus), Sclerocleista (e.g., represented by the species A.ornati), Satoia (e.g., represented by the species A. niger), Neosartorya(e.g., represented by the species A. fumigatus, A. cervinus),Hemicarpenteles (e.g., represented by the species A. clavatus),Petromyces (e.g., represented by the species A. flavus, A. candidus, A.sparsus), Emericella (e.g., represented by the species A. nidulans, A.versicolor, A. ustus), and Fenellia (e.g., represented by the species A.terreus).

In a particular preferred embodiment, the Aspergillus host cell isselected from the group consisting of A. oryzae, A. aculeatus, A.ficuum, A. flavus, A. foetidus, A. soja, A. sake, A. niger, A. nidulansand A. japonicus. Of these, Aspergillus oryzae and Aspergillus niger, A.parasiticus, and A. phoenicus are most preferred.

The above examples of host cells of the invention are named according tothe presently accepted taxonomy.

Toxins

The toxin the production of which is to be reduced or eliminatedaccording to the present invention may be any toxin produced byAspergilli or encoded by a gene harbored in Aspergilli, but notnecessarily expressed. For instance, it is know that aflatoxin genes arepresent in A. oryzae, but not expressed from this species. However, itmay still be advantageous to eliminate aflatoxin pathway genes eventhough they are not expressed. This will be described further below andillustrated in Example 6.

In particular, the toxin to be reduced or eliminated is selected fromthe group consisting of cyclopiazonic acids (CPA), e.g., the alpha orbeta form thereof, kojic acid (KA), 3-nitropropionic acid (NPA), emodin,malformins (e.g., Malformin A or B), aflatoxins, ochratoxins, flaviolin,and secalonic acids (e.g., secalonic D). For a further description ofthese toxins see the Background of the Invention section herein as wellas the Handbook of Toxic Fungal Metabolites referred to in the section.

Dimethylallyl-cyclo-acetoacetyl-L-tryptophan synthase (DCAT-S) of theinvention

The present invention also relates to isolateddimethylallyl-cycloacetoacetyl-L-tryptophan synthases obtained from afilamentous fungus, selected from the group consisting of (a) adimethylallyl-cycloacetoacetyl-L-tryptophan synthase having the aminoacid sequence of SEQ ID NO: 2; (b) an allelic variant of (a); and (c) afragment of (a) or (b), wherein the fragment hasdimethylallyl-cycloacetoacetyl-L-tryptophan synthase activity.

Preferably, the dimethylallyl-cycloacetoacetyl-L-tryptophan synthases ofthe present invention comprise the amino acid sequence of SEQ ID NO: 2,or an allelic variant thereof. In a more preferred embodiment, thedimethylallyl-cycloacetoacetyl-L-tryptophan synthases of the presentinvention comprise the amino acid sequence of SEQ ID NO: 2. In anotherpreferred embodiment, a dimethylallyl-cycloacetoacetyl-L-tryptophansynthase of the present invention has the amino acid sequence of SEQ IDNO: 2 or fragments thereof, wherein the fragment hasdimethylallyl-cycloacetoacetyl-L-tryptophan synthase activity. Afragment of SEQ ID NO: 2 is a polypeptide having one or more amino acidsdeleted from the amino and/or carboxy terminus of this amino acidsequence. In a most preferred embodiment, thedimethylallyl-cycloacetoacetyl-L-tryptophan synthase has the amino acidsequence of SEQ ID NO: 2.

Preferably, a fragment of SEQ ID NO: 2 of at least 320 amino acidresidues, and most preferably at least 350 amino acid residues.

An allelic variant denotes any of two or more alternative forms of agene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in phenotypic polymorphismwithin populations. Gene mutations can be silent (no change in theencoded polypeptide) or may encode polypeptides having altered aminoacid sequences. The term allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

The amino acid sequence of SEQ ID NO: 2 or a partial sequence thereofmay be used to design an oligonucleotide probe, or a nucleic acidsequence encoding a dimethylallyl-cycloacetoacetyl-L-tryptophan synthaseof the present invention, such as the nucleic acid sequence of SEQ IDNO: 1, or a subsequence thereof, may be used to identify and clone DNAencoding dimethylallyl-cycloacetoacetyl-L-tryptophan synthases fromother filamentous fungal strains according to methods well known in theart. In particular, such probes can be used for hybridization with thegenomic or cDNA of the genus or species of interest, following standardSouthern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 15, preferably at least 25,and more preferably at least 40 nucleotides in length. Longer probes canalso be used. Both DNA and RNA probes can be used. The probes aretypically labeled for detecting the corresponding gene (for example,with ³²P, ³H, ³⁵S, biotin, or avidin).

Hybridization indicates that the nucleic acid sequence hybridizes to theoligonucleotide probe corresponding to the polypeptide encoding part ofthe nucleic acid sequence shown in SEQ ID NO: 1 or contained in pJaL499,under low to high stringency conditions (i.e., prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and either 25, 35 or 50% formamide forlow, medium and high stringencies, respectively), following standardSouthern blotting procedures.

Thus, a genomic, cDNA or combinatorial chemical library prepared fromother filamentous fungal strains may be screened for DNA whichhybridizes with the probes described above and which encodes adimethylallyl-cycloacetoacetyl-L-tryptophan synthase. Genomic or otherDNA from other filamentous fungal strains may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA which is homologous with SEQ ID NO: 1,the carrier material is used in a Southern blot in which the carriermaterial is finally washed three times for 30 minutes each using 2×SSC,0.2% SDS preferably at least 50° C., more preferably at least 55° C.,more preferably at least 60° C., more preferably at least 65° C., evenmore preferably at least 70° C., and most preferably at least 75° C.Molecules to which the oligonucleotide probe hybridizes under theseconditions are detected using X-ray film.

In a preferred embodiment, a dimethylallyl-cycloacetoacetyl-L-tryptophansynthase of the present invention is obtained from a strain ofAspergillus, and more preferably from A. oryzae, e.g., the polypeptidewith the amino acid sequence of SEQ ID NO: 2.

As defined herein, an “isolated”dimethylallyl-cycloacetoacetyl-L-tryptophan synthase is a polypeptidewhich is essentially free of other polypeptides, e.g., at least about20% pure, preferably at least about 40% pure, more preferably about 60%pure, even more preferably about 80% pure, most preferably about 90%pure, and even most preferably about 95% pure, as determined bySDS-PAGE.

The present invention also relates to isolated nucleic acid sequencesencoding dimethylallyl-cycloacetoacetyl-L-tryptophan synthases obtainedfrom a filamentous fungus, and in a more preferred embodiment, thenucleic acid sequence is obtained from an Aspergillus sp, e.g., A.oryzae, in particular the nucleic acid sequence set forth in SEQ IDNO: 1. In another more preferred embodiment, the nucleic acid sequenceis the sequence contained in plasmid pJaL499. The present invention alsoencompasses nucleic acid sequences which differ from SEQ ID NO: 1 byvirtue of the degeneracy of the genetic code. The present invention alsorelates to subsequences of SEQ ID NO: 1 or the polypeptide encoding partof pJaL499, which encode polypeptide fragments which havedimethylallyl-cycloacetoacetyl-L-tryptophan synthase activity. Asubsequence of SEQ ID NO: 1 or of the polypeptide encoding part ofpJaL499 is a nucleic acid sequence encompassed by SEQ ID NO: 1 or thepolypeptide encoding part of pJaL499 except that one or more nucleotidesfrom the 5′ and/or 3′ end have been deleted. Preferably, a subsequenceof SEQ ID NO: 1 contains at least 870 nucleotides, more preferably atleast 960 nucleotides, and most preferably at least 1050 nucleotides.

The nucleic acid sequences may be obtained from microorganisms that aretaxonomic equivalents of Aspergillus oryzae.

The techniques used to isolate or clone such nucleic acid sequences aredescribed herein. The term “isolated nucleic acid sequence” as usedherein refers to a nucleic acid sequence which is essentially free ofother nucleic acid sequences, e.g., at least about 20% pure, preferablyat least about 40% pure, more preferably at least about 60% pure, evenmore preferably at least about 80% pure, and most preferably at leastabout 90% pure as determined by agarose electrophoresis. The nucleicacid sequence may be of genomic, cDNA, RNA, semi-synthetic, syntheticorigin, or any combinations thereof.

Modification of a nucleic acid sequence encoding adimethylallyl-cycloacetoacetyl-L-tryptophan synthase of the presentinvention may be necessary for the synthesis of enzymes substantiallysimilar to the polypeptide. The term “substantially similar” to thedimethylallyl-cycloacetoacetyl-L-tryptophan synthase refers tonon-naturally occurring forms of the enzyme. Thesedimethylallyl-cycloacetoacetyl-L-tryptophan synthases may differ in someengineered way from the enzyme isolated from its native source. Forexample, it may be of interest to synthesize variants of the enzymewhere the variants differ in specific activity, thermostability, pHoptimum, or the like using, e.g., site-directed mutagenesis. Theanalogous sequence may be constructed on the basis of the nucleic acidsequence presented as the polypeptide encoding part of SEQ ID NO: 1,e.g., a subsequence thereof, and/or by introduction of nucleotidesubstitutions which do not give rise to another amino acid sequence ofthe polypeptide encoded by the nucleic acid sequence, but whichcorresponds to the codon usage of the host organism intended forproduction of the polypeptide, or by introduction of nucleotidesubstitutions which may give rise to a different amino acid sequence.For a general description of nucleotide substitution, see, e.g., Ford etal., 1991, Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in a biologically activedimethylallyl-cycloacetoacetyl-L-tryptophan synthase. Amino acidresidues essential to the activity of thedimethylallyl-cycloacetoacetyl-L-tryptophan synthase encoded by anisolated nucleic acid sequence of the invention, and thereforepreferably not subject to substitution, may be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, mutations areintroduced at every positively charged residue in the molecule, and theresultant mutant molecules are tested fordimethylallyl-cycloacetoacetyl-L-tryptophan synthase activity toidentify amino acid residues that are critical to the activity of themolecule. Sites of substrate-enzyme interaction can also be determinedby analysis of the three-dimensional structure as determined by suchtechniques as nuclear magnetic resonance analysis, crystallography orphotoaffinity labelling (see, e.g., de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904;Wlodaver et al., 1992, FEBS Letters 309: 59-64).

A preferred use of the nucleic acid sequence of the invention orhomologues or fragments thereof is to eliminate or reduce thedimethylallyl-cycloacetoacetyl-L-tryptophan synthase activity of a givenhost cell, in particular a cell of A. oryzae, thereby reducing oreliminating the production of CPA from said cell.

The present invention also relates to nucleic acid constructs,recombinant expression vectors, and host cells containing the nucleicacid sequence of SEQ ID NO: 1 or the polypeptide encoding part ofpJaL499, subsequences or homologues thereof, for expression of thesequences. The constructs and vectors may be constructed as describedherein. The host cell may be any cell suitable for the expression of thenucleic acid sequence and may be selected e.g., from the parent ormutant cells described herein.

Genetic Modifications of the Host Cell

In order to express significantly reduced levels of one or more toxins,the host cell of the invention is genetically modified which may beachieved by using standard technologies known to the person skilled inthe art. The gene sequences responsible for production of toxin activitymay be inactivated or partially or entirely eliminated. Thus, anAspergillus mutant host cell according to the invention expressesreduced or undetectable levels of one or more toxins.

In a particular embodiment, the inactivation is obtained by modificationof the respective structural or regulatory regions (such as genes)involved in the formation or secretion of the toxin of choice. Known anduseful techniques include, but are not limited to, specific or randommutagenesis, PCR generated mutagenesis, site specific DNA deletion,insertion and/or substitution, gene disruption or gene replacement,anti-sense techniques, or a combination thereof.

Mutagenesis may be performed using a suitable physical or chemicalmutagenizing agent. Examples of a physical or chemical mutagenezingagent suitable for the present purpose include, but are not limited to,UV irradiation, ionizing irradiation such as gamma irradiation,hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methylhydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodiumbisulfite, and nucleotide analogues. When such agents are used themutagenesis is typically performed by incubating the cell to bemutagenized in the presence of the mutagenizing agent of choice undersuitable conditions, and selecting for cells showing a significantlyreduced production of the targeted toxin(s).

Reduction or elimination of the production of a given toxin by a hostcell may also be achieved by modification of a nucleotide sequenceinvolved in or otherwise necessary for the production or secretion ofthe toxin. For instance, the nucleotide sequence may encode a geneproduct having a necessary function in the pathway leading to toxinproduction. Nucleotide sequence may, e.g., be the one shown in SEQ IDNO: 1 or the polypeptide encoding part of pJaL499. Modification may beaccomplished by the introduction, substitution or removal of one or morenucleotides in the nucleotide sequence or a regulatory element requiredfor the transcription or translation of the sequence. For example,nucleotides may be inserted or removed so as to result in theintroduction of a stop codon, the removal of a start codon or a changeof the open reading frame of the nucleotide sequence. The modificationor inactivation of the sequence or a regulatory element thereof may beaccomplished by site-directed or random mutagenesis or PCR generatedmutagenesis in accordance with methods known in the art. Although, inprinciple, the modification may be performed in vivo, i.e., directly onthe cell expressing the toxin gene(s), it is presently preferred thatthe modification be performed in vitro as exemplified below.

An example of a convenient way to inactivate or reduce production of atoxin of interest, e.g., CPA, of a filamentous fungal cell of choice isbased on techniques of gene replacement, gene deletion, or genedisruption. For example, in the gene disruption method, a nucleic acidsequence corresponding to the endogenous gene or gene fragment ofinterest (e.g., the DCAT-S gene of the invention) is mutagenized invitro to produce a defective nucleic acid sequence which is thentransformed into the parent cell to produce a defective gene. Byhomologous recombination, the defective nucleic acid sequence replacesthe endogenous gene or gene fragment. It may be desirable that thedefective gene or gene fragment also encodes a marker, which may be usedfor selection of transformants in which the nucleic acid sequence hasbeen modified or destroyed.

Alternatively, modification or inactivation of the gene may be performedby established anti-sense techniques using a nucleotide sequencecomplementary to the nucleic acid sequence of the gene. Morespecifically, expression of the gene by a filamentous fungal cell may bereduced or eliminated by introducing a nucleotide sequence complementaryto the nucleic acid sequence of the gene, which may be transcribed inthe cell and is capable of hybridizing to the mRNA produced in the cell.Under conditions allowing the complementary anti-sense nucleotidesequence to hybridize to the mRNA, the amount of protein translated isthus reduced or eliminated.

Following mutagenesis or other modification of genes of a toxin pathwaythe mutants are screened for reduced or eliminated toxin production.Specific examples of how to screen for toxins are given in the examplesbelow. Alternatively, useful screening assays are described in the“Handbook of Toxic Fungal Metabolites” or are available at institutionsnormally checking the level of mycotoxins in, e.g., foodstuffs.

Therefore, due to genetic modification, the Aspergillus mutant host cellaccording to the present invention expresses significantly reducedlevels of toxin(s). In a preferred embodiment, the level of thesetoxin(s) expressed by the mutant host cell has been reduced individuallymore than about 50%, preferably more than about 85%, more preferablymore than about 90%, and most preferably more than about 95%, or evenmore than 99%. In another preferred embodiment these toxins in themutant host cell according to the invention may be reduced in anycombination. In a further preferred embodiment, the product expressed bythe host cell is essentially free from at least one toxin of the groupof cyclopiazonic acid, kojic acid, 3-nitropropionic acid, emodin,malformin, aflatoxins, ochratoxins and secalonic acids. In particularlypreferred embodiment, the product expressed by the host cell isessentially free from at least cyclopiazonic acid, more particularlyfree from at least cyclopiazonic acid and kojic acid or an aflatoxin,and most preferably free from at least cyclopiazonic acid, kojic acidand 3-nitropropionic acid.

In a preferred embodiment the host cell is a strain of A. oryzae havinga reduced or eliminated production of one or more of NPA, CPA, kojicacid (KA) or maltoryzin, preferably at least two of these toxins such asNPA and CPA; NPA and KA; CPA and KA; or NPA, CPA and KA. Furthermore, inaddition to the elimination or reduction of one or more of these toxins,preferably a gene of an aflatoxin pathway of choice is inactivated sothat the resulting mutant strain is unable to produce the aflatoxin.Aflatoxin genes from A. flavus are well known and may be used toidentify the corresponding genes in A. oryzae, which can then beinactivated by methods known in the art.

In another preferred embodiment the host cell is a strain of A. niger orA. ficuum having a reduced or eliminated production of one or more ofmalformin (eg malformin Al or B), an ochratoxin (e.g., ochratoxin A),and flaviolin, preferably at least two of these toxins such as malforminand ochratoxin; malformin and flaviolin; ochratoxin and flaviolin; andmalformin, ochratoxin and flaviolin.

In another preferred embodiment the host cell is a strain of A.aculeatus having a reduced or eliminated production of one or more ofone of the secalonic acids (e.g., secalconic acid D) or emodin (aprecursor to secalonic D), preferably of both of these types of toxin.

Methods of Producing Polypeptides

By the method of the present invention, the amount of certain targetedtoxin(s) is significantly reduced, whereas the characteristics of themutant host cell in terms of stable maintenance in the cell of thegenetically modified genes encoding the polypeptide of interest,production capability of the cell and yield of the polypeptide ofinterest is substantially maintained. More specifically, by the methodof the invention, the host cell is genetically modified withinstructural and/or regulatory regions necessary for the production orsecretion of one or more toxins of interest thereby eliminating orreducing the production or secretion of said toxin(s).

Therefore, another aspect of the invention provides a method ofproducing polypeptides or proteins in an Aspergillus mutant host cellaccording to the invention, including heterologous polypeptides orproteins, which method comprises introducing into said mutant host cella nucleic acid sequence encoding the polypeptide of interest,cultivating the mutant host cell in a suitable growth medium, andrecovering said polypeptide of interest.

Thus, the mutant host cell according to the invention must containstructural and regulatory genetic regions necessary for the expressionof the polypeptide of interest. The nature of such structural andregulatory regions depends to a large extent on the product aimed andthe particular Aspergillus host strain. The genetic design of the hostcell according to the invention may be accomplished by the personskilled in the art using standard recombinant DNA technology for thetransformation or transfection of a host cell (see, e.g., Sambrook etal.).

Preferably, the host cell is modified by methods known in the art forthe introduction of an appropriate cloning vehicle, i.e., a plasmid or avector, comprising a DNA fragment encoding the desired polypeptide ofinterest. The cloning vehicle may be introduced into the host celleither as an autonomously replicating plasmid or integrated into thechromosome. Preferably, the cloning vehicle comprises one or morestructural regions operably linked to one or more appropriate regulatoryregions.

The structural regions are regions of nucleotide sequences encoding thepolypeptide of interest. The regulatory regions include promoter regionscomprising transcription and translation control sequences, terminatorregions comprising stop signals, and polyadenylation regions. Thepromoter, i.e., a nucleotide sequence exhibiting a transcriptionalactivity in the host cell of choice, may be one derived from a geneencoding an extracellular or an intracellular protein, preferably anenzyme, such as an amylase, a glucoamylase, a protease, a lipase, acellulase, a xylanase, an oxidoreductase, a pectinase, a cutinase, or aglycolytic enzyme. Examples of suitable promoters for directing thetranscription of the nucleic acid constructs in the methods of thepresent invention are promoters obtained from the genes encodingAspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase,Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stablealpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase(glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulansacetamidase, Aspergillus oryzae acetamidase (amdS), Fusarium oxysporumtrypsin-like protease (U.S. Pat. No. 4,288,627), and mutant, truncated,and hybrid promoters thereof. Particularly preferred promoters are theNA2-tpi promoters (a hybrid of the promoters from the genes encodingAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), glucoamylase, and TAKA amylase promoters.

The cloning vehicle may also include a selectable marker. A selectablemarker is a gene the product of which provides for biocide or viralresistance, resistance to heavy metals, prototrophy to auxotrophs, andthe like. A selectable marker for use in a filamentous fungal host cellmay be selected from the group including, but not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents from otherspecies. Preferred for use in an Aspergillus cell are the amdS and pyrGgenes of Aspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

Furthermore, selection may be accomplished by co-transformation, whereinthe transformation is carried out with a mixture of two vectors and theselection is made for one vector only.

The procedures used to ligate the DNA construct of the invention, thepromoter, terminator and other elements, respectively, and to insertthem into suitable cloning vehicles containing the information necessaryfor replication, are well known to persons skilled in the art (see,e.g., Sambrook et al., 1989; ibid.).

The mutant filamentous fungal cell is cultivated in a nutrient mediumsuitable for production of a polypeptide of interest using methods knownin the art. For example, the cell may be cultivated by shake flaskcultivation, small-scale or large-scale fermentation (includingcontinuous, batch, fed-batch, or solid state fermentations) inlaboratory or industrial fermentors performed in a suitable medium andunder conditions allowing the heterologous polypeptide to be expressedand/or isolated. The cultivation takes place in a suitable nutrientmedium comprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable media are available fromcommercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). The secreted polypeptide can be recovered directly from themedium.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptide. These detection methods may include use ofspecific antibodies, formation of an enzyme product, disappearance of anenzyme substrate, or SDS-PAGE. For example, an enzyme assay may be usedto determine the activity of the polypeptide. Procedures for determiningenzyme activity are known in the art for many enzymes.

The resulting polypeptide may be isolated by methods known in the art.For example, the polypeptide may be isolated from the nutrient medium byconventional procedures including, but not limited to, centrifugation,filtration, extraction, spray drying, evaporation, or precipitation. Theisolated polypeptide may then be further purified by a variety ofprocedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), or extraction (see, e.g., ProteinPurification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, NewYork, 1989).

Products

The desired end product, i.e., the polypeptide of interest expressed bythe Aspergillus mutant host cell, may be any homologous or heterologousprotein or peptide.

The polypeptide may be any polypeptide heterologous to the mutantfilamentous fungal cell. The term “polypeptide” is not meant herein torefer to a specific length of the encoded product and, therefore,encompasses peptides, oligopeptides, and proteins. The heterologouspolypeptide may also be an engineered variant of a polypeptide. The term“heterologous polypeptide” is defined herein as a polypeptide, which isnot native to the filamentous fungal cell. The mutant filamentous fungalcell may contain one or more copies of the nucleic acid sequenceencoding the heterologous polypeptide.

In the methods of the present invention, the mutant filamentous fungalcell may also be used for the recombinant production of polypeptides,which are native to the cell. The native polypeptides may berecombinantly produced by, e.g., placing a gene encoding the polypeptideunder the control of a different promoter to enhance expression of thepolypeptide, to expedite export of a native polypeptide of interestoutside the cell by use of a signal sequence, and to increase the copynumber of a gene encoding the polypeptide normally produced by the cell.The present invention also encompasses, within the scope of the term“heterologous polypeptide”, such recombinant production of homologouspolypeptides, to the extent that such expression involves the use ofgenetic elements not native to the cell, or use of native elements whichhave been manipulated to function in a manner that do not normally occurin the host cell.

In a more specific embodiment, the product is a therapeutically activepeptide or protein, such as a hormone, in particular insulin, growthhormone, glucagon, or somatostatin; an interleukin, in particularinterferon; a haematopoietic growth factor, in particular PDGF (plateletderived growth factor), EPO (erythropoietin), or TPO (thrombopoietin); aprotease, in particular factor VII, factor VIII, urokinase, chymosin, orTPA; or serum albumin.

In another preferred embodiment, the product is an enzyme of fungal orbacterial origin. The enzyme is preferably a glycosidase enzyme, e.g.,an amylase, in particular an alpha-amylase, a beta-amylase or aglucoamylase; a glucan alpha-1,4-glucosidase; an aminopeptidase; acarbohydrase; a carboxypeptidase; a catalase; a cellulase, in particularan beta-1,4-endoglucanase or a beta-1,3(4)-endoglucanase; acellulose-beta-1,4-cellobiosidase; a chitinase; a cutinase; acyclodextrin glycosyltransferase; a deoxyribonuclease; a galactanase; agalactosidase, in particular an alpha-galactosidase or abeta-galactosidase; an endoglucanase, in particular anbeta-1,3-endoglucanase, an alpha-1,3-endoglucanase, anbeta-1,2-endoglucanase, or a beta-1,6-endoglucanase; a glucosidase, inparticular an alpha-glucosidase or a beta-glucosidase; an invertase; alaccase; a lipolytic enzyme, in particular a lipase, an esterase, aphospholipase, or a lyso-phospholipase; a lyase or a pectate lyase; amannase; a mannosidase; a polygalacturonase; a mutanase; an oxidase oran oxidoreductase, such as a peroxidase or a polyphenoloxidase; anoxygenase; a pectinase, an endo-peptidase or an exo-peptidase; aphytase; a polygalacturonase; a protease; a ribonuclease; atransglutaminase; and a xylanase, in particular a beta-1,4-endoxylanaseor a xylan-beta-1,3-endoxylosidase.

In another preferred embodiment the product is a hybrid polypeptide,such as prochymosin and pro-trypsin-like proteases. The heterologousprotein expressed by the host cell may, under suitable conditions, e.g.,absence of substantial protease activities, also be a precursor proteinsuch as a zymogen, a hybrid protein, a protein obtained as a prosequence or a pre-pro sequence, or any other immature form.

The invention is further illustrated with reference to the followingexamples, which should not in any way be construed as limiting the scopeof the invention as defined in the appended claims.

Aspergillus Mutants having Silent Toxin Gene(s) Eliminated

The biosynthtic pathway for aflatoxin has been studied in theaflatoxinogenic species Aspergillus flavus and Aspergillus parasiticus.In both species a number of genes have been identified and shown to mapin a large cluster (reviewed by Woloshuk, C. P. and Prieto, R, FEMSMicrobiology Letter (1998) 160:169-176). Several of the genes, includingaflR, which encodes a gene regulating expression of the other pathwaygenes, and omtA, encoding O-methyltransferase, have been cloned andsequenced.

Aflatoxin genes are present in the genome of A. oryzae, but are notexpressed from this species. Even though no aflatoxin is expressed it isstill advantageous to eliminate one or more of these silent aflatoxinpathway genes. Aspergilli mutants, e.g., A. oryzae mutants, havingaflatoxin genes, such as the aflR and/or omtA genes, eliminated areadvantageous, because then new mutant strains need not be tested forproduction of the aflatoxin(s) in question.

Thus, in an aspect the invention relates to Aspergillus mutant cells,suitable for the expression of heterologous polypeptides, wherein one ormore silent toxin genes have been eliminate.

That the silent toxin gene(s) have been “eliminated” means that gene(s)in question have been changes or removed, e.g., by gene replacement ordisruption techniques well known in the art (see, e.g., Miller et al.,1985, Molecular and Cellular Biology, p. 1714-1721), in a mannerresulting in that said silent gene(s) is(are) not comprised in themutant cell.

The term “silent” toxin gene(s) means that the toxin gene(s) are notexpressed.

The toxin gene(s) may be eliminated by non-revertably detetion ordisruption of all or part of the toxin gene(s).

The term “non-revertably deletion or disruption of all or part of thetoxin gene(s)” means that the toxin gene(s) in question have been eitherremoved or changed in a manner so that said genes do not encode a toxinand cannot naturally mutate back, e.g., during production to a geneencoding a toxin.

Aspergillus cells in question are any of the above described and may beselected from the group consisting of the Aspergillus subgroupsEurotium, Chaetosartorya, Sclerocleista, Satoia, Neosartorya,Hemicarpenteles, Petromyces, Emericella, and Fenellia.

Toxin gene(s) in question encoding one or more toxins include toxinsselected from the group consisting of cyclopiazonic acid, kojic acid,3-nitropropionic acid, emodin, malformin, aflatoxins, ochratoxins andsecalonic acids.

Specifically contemplated are toxin gene(s) encoding an aflatoxin, inparticular toxin gene(s) from the A. oryzae aflatoxin cluster, inparticular selected from the group comprising omtA, aflR, pksA, Nor-1,fas-beta, fas-alpha, vber-1, avnA, ord-2.

The parent Aspergillus cell may be an A. oryzae cell, in particular A.oryzae A1560 (IFO 0417).

Experimental

Materials and Methods

1. Strains

Aspergillus oryzae A1560 is equal to IFO 04177 (see below).

Aspergillus oryzae IFO 4177: available from Institute for Fermentation,Osaka; 17-25 Juso Hammachi 2-Chome Yodogawaku, Osaka, Japan; see also WO98/12300.

JaL228: Aspergillus oryzae strain in which the gene for a neutralmetalloprotease, NpI, is disrupted; the construction of this strain isdescribed in WO 98/12300.

BECh 1: The construction of this CPA negative Aspergillus oryzae strainis described in Example 1.

BECh 2: The construction of this CPA negative and KA negativeAspergillus oryzae strain is described in Example 1.

BECh 3: The construction of this CPA negative and KA negativeAspergillus oryzae strain is described in Example 1.

BZ14: A strain of Aspergillus oryzae A1560 cotransformed with ToC90 andphD450 as described in WO 92/17573.

JaL 250: The construction of this Aspergillus oryzae strain is describedin Example 6.

Deposit:

An E. coli strain containing the plasmid pJaL499 was deposited withDSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,Mascheroder Weg 1b, D-38124 Braunschweig on Jan. 13, 1999, and obtainedthe deposit number DSM 12622.

2. Genes

DMAT-S: This gene codes for dimethylallyl-L-tryptophan synthase, anenzyme involved in the biosynthesis of ergot alkaloid.

DCAT-S: This gene codes for dimethylallyl-cyclo-acetoacetyl-L-tryptophansynthase, an enzyme involved in the biosynthesis of cyclopiazonic acid(CPA).

pyrG: This gene codes for orotidine-5′-phosphate decarboxylase, anenzyme involved in the biosynthesis of uridine.

3. Plasmids

pAHL: This plasmid is described in WO 97/07202.

pCaHj483: This plasmid is described in WO 98/00529.

pCaHj493: This plasmid is described in Example 2.

pJaL335: This plasmid is described in WO 98/12300.

pJaL499: This plasmid is described in Example 4.

4. Media and Solutions

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Screening Medium 1 (Per Liter) Mannitol 30 g Glucose 10 g Succinic acid10 g Casamino acids 3 g KH₂PO₄ 1 g MgSO₄*7H₂O 0.3 g FeSO₄*7H₂O 0.2 g2.6-dichloro-4-aniline 2 ppm agar 20 gFinal pH 5.6, adjusted with 14% NH₄OH.

Cove N Cove Salt solution 50 ml Sorbitol 218 g dextrose 10 g potassiumnitrate 2.02 g Agar 35 g deionized water 1000 ml

COVE Salt Solution (Per Liter) KCl 26 g MgSO₄ 26 g KH₂PO₄ 76 g TraceMetals Sol'n 50 ml CHCl₃ 2 ml

Trace Metals Solution (Per 1 Liter) Na₂B₄O₇*10H₂O 40 mg CuSO₄*5H₂O 400mg FeSO₄*7H₂O 800 mg MnSO₄*2H₂O 800 mg Na₂MoO₄*2H₂O 800 mg ZnSO₄*7H₂O8000 mg

G1-gly yeast extract 18 g Glycerol 87% 24 ml Pluronic PE6100 1 ml Tapwater ad 1000 ml

1/5 MDU-2BP maltose 9 g MgSO₄*7H₂O 0.2 g NaCl 0.2 g K₂SO₄ 0.4 g KH₂PO₄2.4 g Yeast Extract 1.4 g AMG Trace Metals 0.1 ml Pluronic PE6100 0.02ml Deionized Water ad 1000 mlFinal pH 5.0; prior to inoculation, 1.0 ml of 50% urea is added.

MDU-1B (Per 1 Liter) Maltodextrin MD01 45.0 g MgSO₄*7H₂O 1.0 g NaCl 1.0g K₂SO₄ 2.0 g KH₂PO₄ 12.0 g Yeast Extract 7.0 g AMG Trace Metals 0.5 mlPluronic PE6100 1 mlFinal pH 5.0;Prior to inoculation, 1.3 ml of 50% urea/100 ml medium is added.

AMG Trace Metals Solution (Per 1 Liter) FeSO₄*7H₂O 13.9 g MnSO₄*H₂O 8.45g ZnCl₂ 6.8 g CUSO₄*5H₂O 2.5 g NiCl₂*6H₂O 2.5 g Citric acid ≧3.0 g TraceMetal Solution 1 ml

KM2 medium (Per 1 Liter) Yeast extract 2.5 g K₂HPO₄ 10 g MgSO₄*7H₂O 0.5g KCl 0.5 g FeSO₄ 0.01 g glucose 100 g Final pH adjusted to 6.0For use in solid plates, KMZ medium is solidified with agar, 20 g/l.Triton X-lOO at 300 microliters/l is added as a colony growthrestriction agent.

Nakamura Medium (Per 1 Liter) Sucrose 50 g Peptone 20 g KH₂PO₄ 5 gCaHPO₄ 2.5 g MgSO₄ 2.5 gFinal pH is adjusted to 6.45. AssaysA. Assay Procedure for CPA by HP Capillary Electrophoresis

One ml aliquot of sample is prepared for capillary electrophoresis (CE)analysis by solid phase extraction on a Supelclean LC-18 SPE tube(prepacked 3 ml column from Supelco, cat. no. 5-7012, conditioned with 2ml methanol and 2 ml Milli-Q water). A suction manifold is used in orderto force the fluids through the column. After washing with 3 ml Milli-Qwater, the sample is eluted with 3 ml methanol. Without furthertreatment, eluate is then subjected to CE analysis. Occasionally aprecipitate forms which is removed by centrifugation.

The CE analysis is performed using a Hewlet-Packard photo-diode arrayCE-apparatus (3D-CE). The sample is injected under hydrostatic pressureof 34 mbar over 10 sec. A 50 mm capillary (56 cm effective length) at30° C. is used which has been conditioned with 0.1 M NaOH for 1 min,followed by 100 mM borate/NaOH pH 9.1 for 5 min. The voltage is set at17 kV. UV-spectra are always collected to identify the peak, but the runis followed at 280 nm. A commercial cyclopiazonic acid is used as astandard (Sigma Co., St. Louis Mo., USA, catalog no. C1530, minimum 98%pure). The lower limit of sensitivity of the method is approximately 1ppm.

B. Assay Procedures for CPA by Thin Layer Chromatography (TLC)

1. Analysis of Plate Cultures

Agar plugs are analyzed as described in Filtenborg O., Frisvad J. C. andSvendsen J. A.: “Simple Screening Method for Mold ProducingIntracellular Mycotoxins in Pure Cultures”, Applied and EnvironmentalMicrobiology (1983) 45:581-585.

2. Analysis of Liquid Cultures

10 microliter samples of supernatant are applied at both opposite edgesof a TLC plate (Merck Silica Gel 60). Aliquots of cyclopiazonic acid,Sigma C 1530, dissolved and diluted in a mixture of methanol: chloroform(1:2 by volume) to 50 ppm, 25 ppm, 5 ppm and 2.5 ppm are used asstandards. The plate is first developed in CAP (chloroform: acetone:propan-2-ol =85:15:20 by volume) for 15 minutes, allowed to dry, thenturned around and the other half is subsequently developed in TEF(toluene: ethyl acetate: formic acid=5:4:1 by volume) for 15 minutes.

Alternatively the plate is developed in EMA (ethyl acetate: methanol:25% ammonium hydroxide=16:8:2 by volume) and TEF each for 15 minutes asdescribed above.

The plate is allowed to dry thoroughly (1 hour) in a fume hood, beforespraying with Ehrlich reagent (2 g of 4-dimethylaminobenzaldehyde in 85ml 96% ethanol to which 15 ml 37% hydrochloric acid is subsequentlyadded).

CPA is seen as bluish-violet mushroom shaped spots with a typical lowmigration in the CAP system (a neutral system) whereas the acidic TEFsystem yields a typical prolonged smear midway between the applicationsite and the front of developer. In the EMA system (basic/alkalinesystem) the cyclopiazonic acid is focused to small dense spots.

By direct visual inspection of the plates, CPA concentrations≧2.5 ppmcan be seen as purple zones smears or spots (depending on thedevelopment system). By scanning the TLC plate on a desktop flatbedscanner and then processing and enhancing the electronic image in asuitable Image Processing Programme (in this case Paint Shop Pro 4) thesensitivity is improved with a factor 5 to 10.

The overall sensitivity of this analysis is (without extraction) approx.0.5-1 ppm CPA; using extracts improves the sensitivity at least 10-fold.

C. Assay Procedure for kojic acid by Capillary Electrophoresis

An aliquot of 1 to 3 ml of sample is prepared for CE analysis by solidphase extraction on a Supelclean LC-18 SPE tube (prepacked 3 ml columnfrom Supelco, cat. no. 5-7012), conditioned with methanol followed by 10mM borate/NaOH, 4 M KCl pH 9.1. A suction manifold is used in order toforce the fluids through the column. After washing with 3 ml of 10 mMborate/NaOH, 4 KCl pH 9.1 and 0.3 ml 10 mM borate/NaOH pH 9.1, thesample is eluted with 7.5 ml 10 mM borate/NaOH pH 9.1. Without furthertreatment, eluate is then subjected to CE analysis, following theprocedure described above for cyclopiazonic acid. Occasionally aprecipitate forms which is removed by centrifugation. The lower limit ofsensitivity of the method is approximately 6 ppm.

D. Assay Procedure for kojic acid by Thin Layer Chromatography

An aliquot of sample is applied to the opposing sides of TLC plates asdescribed above for CPA and developed using the same solvent systems asfor CPA. The dried plates are sprayed with 1% FeCl₃ in 0.1 M HCl. Thepresence of kojic acid in the sample is indicated by a red spot andcompared to the intensity of the red spot produced by pure kojic acidapplied as a control. The lower limit of detection is 50 ppm.

E. Assay Procedure for 3-NPA by Capillary Electrophoresis

The need for sample purification preparatory to capillaryelectrophoresis (CE) analysis depends on the conductivity of the sample.If the conductivity is less than 10 mS, the sample is purified by ionexchange over a Varian SAX anion exchanger (Varian Instruments, PaloAlto Calif.) using 0.1 M KCl as the elution buffer. If the conductivityis greater than 100 mS, the sample is extracted using 2-butanolextraction, in which a 2 ml sample is extracted with 6 ml butanol afterprecipitation with acidification/high salt treatment and redissolvingthe precipitate in 10 mM Tris/HCl pH 7.0.

HP-CE apparatus, diode array detection, using a capillary of uncoatedsilica at 50 micro-m and an effective length of 56 cm at a temperatureof 30° C. and a conditioning buffer of 25 mM borate/phosphate pH 7.6 areapplied. The sample is injected under hydrodynamic pressure over a 20sec. period. The voltage is set at 30 kV. The lower limit of sensitivityof the method is 6 ppm.

F. Assay Procedure for 3-NPA by Thin Layer Chromatography

Spots of fermentation liquid are applied to TLC plates and developed asdescribed for CPA. They are then sprayed with diazotized p-nitroanilineas described by W. Majak and R. J. Bose, “Chromatographic methods forthe isolation of miserotoxin and the detection of aliphatic nitrocompounds”, Phytochemistry (1974) 13:1005-1010. The intensity andposition of the spots relative to the control substance are a measure ofthe 3-NPA concentrations. Detection level on TLC plates: 25-50 ppm.

Alternatively, 3-NPA is analyzed spectrophotometrically (λ=540 nm) byadding 50 microliters 1 M NaOH and 70 microliters diazotizedp-nitroaniline to 100 microlter sample. Detection level 5-10 ppm.

EXAMPLE 1

A. Construction of a CPA Negative Strain Derived from A. oryzae Bz 14

Lyophilized spores of a strain of Aspergillus oryzae BZ14 strain weregamma-irradiated at an optimum dose range of between 1000 Gy-1250 Gy,then plated on plates of Screening medium 1 in densities of 25-50colonies/9 cm plate. Colonies producing cyclopiazonic acid form a redreverse side (the underside of the colony) on Screening 1 medium due toa red insoluble CPA-Fe complex.

Approximately 50,000 colonies from the irradiated spores were screenedand 154 CPA deficient colonies, characterized by a creamy/whitishappearance, were isolated. Following re-isolation, 64 strains retained anon-red reverse on plates of Screening medium 1. CPA was not detected in52 strains by the TLC-plug assay. These strains were then cultured inMDU-1B medium under toxin provoking (5 days at 34° C., 250 rpm) shakeflask fermentation conditions. Thirty-six strains presented nodetectable levels of CPA in the supernatant as measured by TLC.

B. Construction of a CPA Negative Strain, BECh 1, Derived from A. oryzaeJaL228

Lyophilized spores of JaL228 were gamma-irradiated and screened asdescribed above. Putative CPA free isolates were grown in shakeflasks onMDU-1B medium under toxin provoking conditions (5 days at 34° C., 250rpm) and supernatants tested for CPA with the TLC method. Thesupernatants were tested as they were or as extracts.

For the extraction 50 ml of the whole sample was acidified with 10 ml0.1 M HCl. This mixture was then vigorously shaken for 3-5 minutes with70 ml methanol/chloroform (1:2). Following phase separation (approx. 3hours), the bottom phase (approximately 25 ml) was transferred to a 300ml beaker and the chloroform allowed to evaporate. The residue wasredissolved in 5 ml chloroform, transferred to a 25 ml beaker and thechloroform evaporated. The residue was dissolved in 100 microliterschloroform.

Ten microliters of supernatant or chloroform extract were applied to theopposite edges of 20 cm×20 cm TLC plates and processed as described inthe previous chapter on the assays.

Three strains (isolates) including BECh 1 did not produce CPA.

C. Construction of CPA Negative and KA Negative Strains, BECh 2 and BECh3

BECh 1 was grown on a Cove N slant. Spores were suspended in 0.01% Tweento a density of 3-5×10⁶ and subjected to short-wave UV irradiation (254nm from germicidal lamp). Spores irradiated with UV doses resulting in1-5% survival were used in the subsequent screening.

Irradiated spores were diluted in KM2 medium to approx. 0.7 spore/100microliters, and 100 microliters were inoculated into each well in 96well microtiter plates. The cultures were incubated statically in amoist chamber at 34° C. for 5-7 days. Then 40 micro-I 1% FeCl₃ in 0.1 MHCl was added to each well with signs of growth.

The emergence of a strong red color indicated kojic acid (KA)production; absence of color indicated a colony deficient in kojic acidproduction.

Alternatively, spores were plated on solidified KM2 (restricted growthwith Triton x-100) and when mature colonies were seen the plates wereflooded with 1% FeCl₃ in 0.1 M HC1. Absence of red zones around coloniesindicate putative non-kojic acid producers.

One hundred thirty-two putative KA free colonies, i.e., those giving nocolor reaction with FeCl₃, were isolated from among approximately 7000microtiter cultures. However, when tested on the primary KM2 screeningagar plates, none of the colonies were confirmed to be KA negative.

Subsequent re-testing in liquid medium under KA provoking conditions ofthe negative colonies on both solid medium and in static liquid KM2medium (30°) narrowed the number of KA negative mutants down to 11. Whenthese strains were tested in shake flasks, KA was produced by eight ofthe strains. The remaining three gave no color reaction on an assaydirectly on supernatant nor when checked by TLC. As expected, KA wasproduced by the control strain BECh 1, when grown in simultaneousparallel cultures. One of the isolated strains exhibited an aberrantmorphology. The remaining two isolates were retested for both CPA and KAafter prolonged growth on MDU-1B. Neither CPA nor KA was detected. Thetwo strains were named BECh 2 and BECh 3.

D. Construction of a 3-NPA Negative A. oryzae Strain which already isCPA Negative and KA Negative

Strain BECh 2 and BECh 3, respectively, are subjected to UV mutagenesisas described above. The irradiated spores are diluted to 0.7 livespore/100 microliters Nakamura medium in 96 well microtiter plates.Incubate for 5-7 days in moist chamber, 30° C.

Fermentation broth samples from wells with growth are either transferredto a new microwell plate and analyzed spectrophotometrically or appliedto TLC plates. Strains negative for 3-NPA are recultivated inshakeflasks with Nakamura medium and analyzed for 3-NPA. Strains stillnegative for 3-NPA are transformed with pCaHj 493 as described inExample 2 and the transformants are treated as described in Example 3.

EXAMPLE 2

Expression of Lipase Gene in A. oryzae Strains JAL228 and BECh 1

A. Construction of plasmid pCaHj493

The lipase plasmid pAHL (WO 97/07202) was digested with BamHI and SalI,and the resulting 916 bp fragment encoding the lipase was isolated.

pCaHj 483, as described in WO 98/00529, was digested with BamHI andXhoI, and the 6757 bp vector fragment was ligated to the lipasefragment. The ligation mixture was used to transform E. coli DH 5αcells, and a transformant harboring the expected plasmid was isolated.The plasmid was termed pCaHj 493.

B. Transformation of pCaHj 493 into JaL228 and BECh-1

Aspergillus oryzae strains JaL228 and BECh1 were transformed withpCaHj493 using selection on acetamide as described in EP-A-0531372.Transformants were spore reisolated twice. Spores from a secondreisolation of each transformant were tested for lipase production inshake flasks and microtiter dish cultures.

EXAMPLE 3

A. Lipase Production in a CPA Negative and a CPA Positive A. oryzaestrain

Eighteen JaL228 transformants and 30 BECh 1 transformants, prepared asdescribed in Example 2, were tested for the production of lipase inshake flask cultures.

Cove N slants of the transformants were harvested using 10 ml of a 0.1%Tween solution, and the spore suspension was used as the inoculum in 100ml of G1-Gly medium in 500 ml two-baffled shake flasks. The cultureswere incubated on a rotary shaker at 250 rpm, 34° C. for 24 hours. Then10 ml of the G1-Gly culture was transferred to 100 ml 1/5MDU-2BP in 500ml shake flasks and incubated further at 34° C., 250 rpm.

Samples were taken after 50 hours, filtered through Miracloth andcentrifuged (4000×g). Lipase concentrations (expressed in LU/ml) in thesupernants were detected using the Single radial immunodiffusion method(Scand. J. Immunol. Vol. 17, suppl. 10, 41-56, (1983), “Handbook ofImmunoprecipitation-in-Gel Techniques”, N. H. Axelsen, ed., BlackwellScientific Publications, 1983).

The 30 CPA negative BECh 1 transformants had lipase yields as high as orbetter than the 18 JaL228 CPA positive transformants. Table 2 belowgives an overview of the distributions.

B. Xylanase Production by CPA Negative and CPA Positive A. oryzaestrains

Ten strains (prepared in Example 1A) with no detectable CPA productionand three strains in which CPA was detectable were evaluated forproduction of xylanase. The results are summarized in Table 1 below.Column 2 shows the amount of xylanase, measured in fungal xylanase units(FXU) as assayed by the Single radial immunodiffusion method (Scand. J.Immunol. Vol 17, suppl. 10, 41-56, 1983, Handbook ofImmunoprecipitation-in-Gel Techniques, N. H. Axelsen, ed., BlackwellScientific Publications, produced in shake flask cultures.

The results show that the CPA negative strains can produce xylanase inamounts comparable to CPA positive strains. TABLE 1 Strain FXU CPA/ppm2-5 360 <2  3-34 440 <2 4-2 550 <2 5-1 250 <2 5-2 475 <2 7-1 530 <2 7-2475 <2 9-5 370 <2 10-4  300 <2 12-2  365 <2 1-1 600 2-3 1-3 650 20 3-1635 7<2 = below detection limitC. Lipase Production in CPA Negative and KA Negative Strains

The two CPA and KA free A. oryzae strains BECh 2 and BECh 3 weretransformed with the plasmid pCaHj 493 as described in Example 2.Transformants were spore isolated twice. Spores from the secondre-isolation of each transformant were tested for lipase production asdescribed in Example 2.

Table 2 shows the frequency distributions of the lipase yields oftransformants from these two strains. The results show that nodeterioration in expression potential occurred as compared to the valuesgiven for the A. oryzae strains BECh 1 and JaL228.

From the same spore suspension used for the lipase production cultures,MDU1B shakeflasks for CPA production were inoculated and incubated for 5days as described previously. DPA analysis was done according to section5B2. None of the BECh1 strains produced CPA whereas 17 of the 18 JaL228strains gave more than 25 ppm, the majority more than 100 ppm CPA. TABLE2 Std. Median Mean Minimum Maximum Dev. Strain N LU/ml LU/ml LU/ml LU/mlLU/ml BECh2 50 3101 3203 350 6329 1490 BECh3 47 2619 2662 203 5455 1057JaL228 18 1890 2330 635 4664 1361 BECh1 30 3025 2733 876 4097 879

EXAMPLE 4

Identification and Genomic Cloning of the A. oryzae DCAT-S Gene

A. Identification of the A. oryzaedimethylallyl-cycloacetoacetyl-L-tryptophan synthase (DCAT-S) Gene

The cDNA clone (pJaL499) harbors the DNA sequence shown in SEQ ID NO: 1,which has been identified to be involved in the CPA biosynthesis by itshomology to a dimethylallyltryptophan synthase (DMAT-S) from Clavicepspurpurea. Sequencing of the A. oryzae cDNA clone showed that it was 1393base pairs in length (SEQ. ID. NO: 1) and encoded an 473 amino acidpolypeptide (SEQ. ID. NO: 2) that was 42.1% identical to the DMAT-S fromClaviceps purpurea.

The A. oryzae DCAT-S polypeptide is involved in the synthesis ofbeta-CPA from cyclo-acetoacetyl-L-tryptophan anddimethylallylpyrophosphate, Nethling D. C. and R. M. McGrath, Can. J.Microbiol. (1977) 23:856-872.

Chromosomal DNA from strains JaL228 and BECh 1 was prepared. The DNA wasdigested with BgII1, Nco1, Xho1 and Spe1 and analyzed by Southernblotting, using the 1 kb ³²P-labelled BglII DNA fragment from pJaL499containing the DCAT-S gene as a probe. Southern blot analysis showedthat the CPA producing strain JaL228 has one DCAT-S gene, whereas inBECh 1 the DCAT-S gene had been deleted from the chromosome.

B. Cloning of a Genomic Clone of the DCAT-S Gene

Genomic restriction mapping of the A. oryzae DCAT-S gene is carried outwith the following restriction enzymes: EcoRI, SaI1, Bbu1, Xho1, andXbaI, using the 1 kb ³²P-labelled DNA BglII fragment from pJaL499containing the DCAT-S gene as a probe (FIG. 1). This shows that there isonly one copy of the DCAT-S gene.

Genomic DNA of JaL228 is partially digested with either Tsp509I or runon a 0.7% agarose gel. Fragments with a size between 7 and 10 kb arepurified.

The purified DNA is then cloned into Lambda ZAP II using protocolsprovided by the manufacturer (Stragtagene®). In vivo excision andrecircularisation of any clone insert contained within the lambda vectorto form a phagemid containing the cloned insert is done for the DNAlibraries, according to instructions provided by the manufacturer.Screening for clones encoding the DCAT-S gene is performed by colonyhybridization using the 1 kb ³²P-labelled DNA Bgl11 fragment frompJaL499 containing the DCAT-S gene as a probe, as outlined in standardmethodology textbooks (e.g., J. Sambrook, E. F. Fritsch, and T.Maniatis, eds. (1989) “Molecular Cloning: A Laboratory Manual”, SecondEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

EXAMPLE 5

Generation of an Aspergillus oryzae CPA Negative Strain by GenomicDisruption of the Aspergillus oryzaedimethylallyl-cycloacetoacetyl-L-tryptophan synthase (DCAT-S)

The DCAT-S gene is disrupted by the one-step gene replacement method (B.L. Miller et al., Mol. Cell. Biol. (1985) 5:1714-1721, and G. May, inApplied Molecular Genetics of Filamentous Fungi, pp. 1-25; J. R.Kinghorn and G. Turner, eds.; Blakie Academic and Professional, 1992) ina pyrG minus strain of A. oryzae, using the A. oryzae pyrG gene as aselection marker.

A. Construction of the DCAT-S Disruption Plasmid

Plasmid pJaL499 is digested with Sac11 and treated with Klenowpolymerase to create blunt ends, and with bacterial alkaline phosphataseaccording to instructions of the manufacturer (Boehringer Mannheim) toremove the 5′ phosphate groups, and then phenol extracted andprecipitated.

Plasmid pJaL335, described in WO 98/12300, is digested with HindIII toobtain a 3.5 kb fragment comprising the A. oryzae pyrG gene, treatedwith Klenow polymerase to create blunt ends, isolated by gelelectrophoresis, and purified. The two fragments were then mixedtogether and ligated. After transformation into E. coli, the coloniescarrying the correct plasmid were identified by restriction enzymedigestion of mini-plasmid preparations. The construction of thedisruption plasmid (pDCAT-S-pyrG) is summarized in FIG. 2.

B. Isolation of a pyrG Minus A. oryzae Strain, JaL250

The A. oryzae strain JaL228 is screened for resistance to5-fluoro-orotic acid to identify spontaneous pyrG mutants. One strain,named JaL250, is identified as being pyrG minus. The mutant is uridinedependent, therefore it can be transformed with the wild type pyrG geneand transformants are selected by the ability to grow in the absence ofuridine.

C. Construction of an Aspergillus oryzae DCAT-S Minus Strain

The 4.9 kb Not1-EcoRI fragment of the plasmid pDCAT-S-pyrG is gelpurified and used to transform the A. oryzae strain JaL250 as describedby Christensen et al., Biotechnology (1988) 6:1419-1422. Transformantsare then selected by their ability to grow in the absence of uridine.After reisolation twice the transformants are screened for their abilityto produce CPA, as described in Example 1.

In order to confirm that the DCAT-S gene is disrupted chromosomal DNA isprepared from transformants that do not produce CPA. The DNA is digestedwith EcoR1 and analyzed by Southern blotting, using the 1 kb ³²Plabelled DNA Bgl11 fragment from pJaL499 containing the DCAT-S gene as aprobe. Transformants that carry a disruption of the DCAT-S gene arerecognized by the shift of the wild type EcoRI band on 6.3 kb to a EcoR1band on 9.8 kb.

EXAMPLE 6

Confirmation that BECh1 and BECh2 Lack Two Genes from the aflatoxinBiosynthetic Pathway Cluster

The presence of the aflR and omtA aflatoxin genes from the s aflatoxinbiosynthetic pathway cluster in A. oryzae IF04177 and a number ofderivatives thereof have been looked for. The aflR homologue from A.oryzae IF04177 was isolated by PCR from genomic DNA with the primers5956 (5′-GGATCCAGGGCTCCCTGGAG-3′) (SEQ ID NO: 3) and 5955(5′-CCTGACCAGCCAGATCTCCT-3′) (SEQ ID NO: 4). A 0.9 kb PCR fragment wasobtained and cloned into the vector pCR2 from Invitrogen. The identityof the cloned fragment was confirmed by sequencing the resultingplasmid, pToC280, with the M13 forward (−40) and reverse primers. TheomtA homologue was also isolated from genomic IF04177 DNA by PCR withthe primers 6120 (5′-AGTGAGAGAACTCCCTCCTC-3′) (SEQ ID NO: 5) and 6121(5′-CCATATCTTCTCAGTCTCCA-3′) (SEQ ID NO: 6). A 1.2 kb fragment wasobtained and cloned into the vector pCR2 from Invitrogen and theresulting plasmid, pToC276, was sequenced with the M13 forward (−40) andreverse primers to confirm the identity of the cloned fragment.

The cloned fragments of aflR and omtA were used as 32P-labelled probesin a hybridization experiment. Genomic DNA from IFO4177, JaL228, BECh1and BECh2 were digested with the restriction enzyme ecoRI and thegenerated fragments were separated on a 0.7% agarose gel. The DNA wasblotted onto a membrane and hybridized under stringent conditions withthe two ³²P labelled probes one at a time (methods are described in J.Sambrook, E. F. Fritsch, T. Maniatis, eds (1989) “Molecular Cloning: ALaboratory Manual”, Second ed., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.). The blot showed positive hybridizationsignals with both probes from IFO4177 and JaL228, while no bands werevisible in the lanes containing BECh1 and BECh2 DNA. In the IFO4177 andJaL228 lanes an approximately 3.8 kb fragment could be seen with theomtA probe and two bands of approximately 0.5 and 4.3 kb could be seenwith the aflR probe.

Consequently, A. oryzae IF04177 contains at least two genes from theaflatoxin biosynthetic pathway, namely the aflR and the omtA genes. InA. flavus and A. parasiticus the two genes are separated byapproximately 32 kb (Woloshuk, C. P. and Prieto, R, FEMS MicrobiologyLetter (1998) 160:169-176). None of these genes are present in theIF04177 derivatives BECh1 and BECh 2.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1-34. (canceled)
 35. A mutant Aspergillus cell suitable for theexpression of heterologous polypeptides, wherein one or more silenttoxin genes have been eliminated.
 36. The mutant of claim 35, whereinone or more toxin gene(s) have been eliminated by non-revertablydetetion or disruption of all or part of the toxin gene(s).
 37. Themutant of claim 35, wherein the Aspergillus cell is selected from thegroup consisting of the Aspergillus subgroups Eurotium, Chaetosartorya,Sclerocleista, Satoia, Neosartorya, Hemicarpenteles, Petromyces,Emericella, and Fenellia.
 38. The mutant of claim 35, wherein the toxingene(s) encode(s) one or more toxins selected from the group consistingof cyclopiazonic acid, kojic acid, 3-nitropropionic acid, emodin,malformin, aflatoxins, ochratoxins and secalonic acids.
 39. The mutantof claim 35, wherein the toxin gene encodes an aflatoxin.
 40. The mutantof claim 35, wherein the toxin gene(s) are from the A. oryzae aflatoxincluster, in particular selected from the group comprising omtA, aflR,pksA, Nor-1, fas-beta, fas-alpha, vber-1, avnA, ord-2.
 41. The mutant ofclaim 40, wherein the parent Aspergillus cell is an A. oryzae, inparticular A. oryzae A1560 (IFO 0417).
 42. The mutant of claim 41,wherein the aflatoxin gene(s) are selected from the group consisting ofomtA and aflR.
 43. A toxin-deficient Aspergillus mutant host cell usefulfor the production of a heterologous polypeptide of interest, which cellhas been genetically modified in order to produce less of at least onetoxin as compared to an Aspergillus parental cell, when cultured underthe same conditions.
 44. The mutant cell of claim 43, which comprises afirst nucleic acid sequence encoding the polypeptide of interest and asecond nucleic acid sequence comprising a modification of at least oneof the genes responsible for the biosynthesis or secretion of the toxin.45. The mutant cell of claim 43, wherein the cell comprises at least twocopies of the first nucleic acid sequence.
 46. The mutant cell of claim43, wherein the polypeptide of interest is native to the mutant cell.47. The mutant cell of claim 43, wherein the polypeptide of interest isheterologous to the mutant cell.
 48. The mutant cell of claim 43,wherein the toxin is selected from the group consisting of cyclopiazonicacid, kojic acid, 3-nitropropionic acid, emodin, malformin, aflatoxins,ochratoxins and secalonic acids.
 49. The mutant cell of claim 43,wherein the toxins include a first toxin which is cyclopiazonic acid anda second toxin which is selected from the group of kojic acid,3-nitropropionic acid and aflatoxins.